Schneider PN et al. (2011), Use of fully modified 2''-O-methyl antisense ol...

XB-ART-43002

Genesis March 1, 2011; 49 (3): 117-23.

Use of fully modified 2''-O-methyl antisense oligos for loss-of-function studies in vertebrate embryos.

Schneider PN , Olthoff JT , Matthews AJ , Houston DW .

Abstract
Antisense oligonucleotides are commonly employed to study the roles of genes in development. Although morpholino phosphorodiamidate oligonucleotides (morpholinos) are widely used to block translation or splicing of target gene products'' the usefulness of other modifications in mediating RNase-H independent inhibition of gene activity in embryos has not been investigated. In this study, we investigated the extent that fully modified 2''-O-methyl oligonucleotides (2''-OMe oligos) that can function as translation inhibiting reagents in vivo, using Xenopus and zebrafish embryos. We find that oligos against Xenopus β-catenin, wnt11, and bmp4 and against zebrafish chordin (chd), which can efficiently and specifically generate embryonic loss-of-function phenotypes comparable with morpholino injection and other methods. These results show that fully modified 2''-OMe oligos can function as RNase-H independent antisense reagents in vertebrate embryos and can thus serve as an alternative modification to morpholinos in some cases.

PubMed ID: 21442720
PMC ID: PMC3121920
Article link: Genesis
Grant support: [+]
Genes referenced: bmp4 cat.2 chrd.1 gsc nodal3.1 otx2 pax8 sia1 szl wnt11
Morpholinos: bmp4 MO1 ctnnb1 MO1 wnt11b MO1

Article Images: [+] show captions

	Figure 1. 2′-O-Methyl oligos against Xenopus β-catenin inhibit dorsal axis formation and β-catenin signaling. (a) Phenotypes of uninjected embryos (stage 31/32); 30/30, 100% normal phenotype (two experiments). Anterior is to the left in all cases. (b) Phenotypes of embryos injected with 6 ng β-cat-2ome oligo; 24/27, 89% axis deficiency (two experiments). (c) Quantitative real-time PCR of dorsal (gsc, sia and nr3) markers, ventral markers (szl) and β-catenin (β-cat) in control (Un) and 6 ng β-cat-2ome oligo-injected (6ng 2ome) embryos at stage 10.25. (c′) Average (mean) relative expression levels of nr3 in controls and in 6 ng β-cat-2ome oligo-injected embryos from six separate experiments (including those in Figs. 1c and 3d). Error bars indicate standard deviation; P-value by t-test was ∼0.0001. (d) Immunoblotting analysis of β-catenin protein levels (top panel) and α-tubulin (lower level) in uninjected control (Un) and in 6 ng β-cat-2ome oligo-injected embryos at late blastula, early gastrula, and early neurula stages. The relative abundance of β-catenin compared with tubulin is shown below each lane. Reproduced with permission of the Publisher, John Wiley & Sons.
	Figure 2. Comparison of 2′-O-methyl oligo and morpholino oligo activity. (a) Phenotypes of uninjected, β-cat morpholino-injected and β-cat-2ome oligo-injected embryos, 8 ng and 4 ng doses (stage 28). Un, 18/18, 100% normal; 8 ng MO, 8/16, 50% axis deficiency; 4 ng MO, 0/10, 0% axis deficiency; 8 ng 2ome, 14/14, 100% axis deficiency, 4 ng 2ome, 9/17, 53% axis deficiency. (b) Quantitative real-time PCR of nr3 in control (Un) and oligo-injected embryos at stage 10.25. Oligo doses are 8 ng, 6 ng, and 3 ng. (c) Immunoblotting analysis of β-catenin protein levels (top panel) and α-tubulin (lower level) in uninjected control (Un) and in β-cat-MO and 2ome oligo-injected embryos at the late blastula stages (8 ng, 6 ng, and 3 ng). The relative abundance of β-catenin compared with tubulin is shown below each lane. Reproduced with permission of the Publisher, John Wiley & Sons.
	Figure 3. Specificity of 2′-O-methyl oligos against β-catenin. (a) Phenotypes of uninjected embryos (stage 17); 20/20, 100% normal phenotype. (b) Phenotypes of embryos injected with 6 ng β-cat-2ome oligo; 15/15, 100% axis deficiency. (c) Phenotypes of embryos coinjected with 6 ng β-cat-2ome oligo and 50 pg β-catenin mRNA (dorsal injection); 3/15, 20% axis deficiency. (d) Quantitative real-time PCR of dorsal and ventral markers in control (Un) and oligo-injected embryos at stage 10.25. (e) Quantitative real-time PCR of anterodorsal (otx2, myod) and ventroposterior (szl, evx1) markers in control (Un) and oligo-injected embryos at stage 14. Reproduced with permission of the Publisher, John Wiley & Sons.
	Figure 4. 2′-O-methyl oligos against Xenopus wnt11 and bmp4 cause pronephric and tail fin/proctodeal defects. (a) pax8 expression in an uninjected control embryo (stage 24/25); 20/20, 100% normal expression (one experiments). (b) pax8 expression in embryos injected unilaterally with 20 ng of wnt11-MO; 5/9, 56% reduced pronephros (one experiment). (c) pax8 expression in embryos injected unilaterally with 6 ng of wnt11-2ome; 6/8, 75% reduced pronephros (one experiment). (d) Phenotypes of uninjected embryos (stage 36/7); 40/40, 100% normal phenotype (two experiments). (e) Phenotypes of embryos injected with 4ng bmp4-2ome oligo. Arrows indicate ventral tail fin (solid line) and proctodeal (dashed line) abnormalities; 16/28, 57% tail defects (two experiments). Reproduced with permission of the Publisher, John Wiley & Sons.

References [+] :

Boiziau, Inhibition of translation initiation by antisense oligonucleotides via an RNase-H independent mechanism. 1991, Pubmed, Xenbase